EP2289900A1 - Bisphosphonates en tant qu'inhibiteurs d'acide sphingomyélinase - Google Patents

Bisphosphonates en tant qu'inhibiteurs d'acide sphingomyélinase Download PDF

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EP2289900A1
EP2289900A1 EP09168712A EP09168712A EP2289900A1 EP 2289900 A1 EP2289900 A1 EP 2289900A1 EP 09168712 A EP09168712 A EP 09168712A EP 09168712 A EP09168712 A EP 09168712A EP 2289900 A1 EP2289900 A1 EP 2289900A1
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compound
cystic fibrosis
independently
asmase
diseases
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Christoph Arenz
Gundula Roth
Stefan Uhlig
Daniela Drescher
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Humboldt Universitaet zu Berlin
Rheinisch Westlische Technische Hochschuke RWTH
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Humboldt Universitaet zu Berlin
Rheinisch Westlische Technische Hochschuke RWTH
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Priority to EP09168712A priority Critical patent/EP2289900A1/fr
Priority to PCT/EP2010/062134 priority patent/WO2011023624A1/fr
Priority to EP10759827A priority patent/EP2470550A1/fr
Priority to US13/391,328 priority patent/US20120178720A1/en
Priority to CA2771933A priority patent/CA2771933A1/fr
Publication of EP2289900A1 publication Critical patent/EP2289900A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C07F9/3843—Polyphosphonic acids containing no further substituents than -PO3H2 groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3808—Acyclic saturated acids which can have further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C07F9/386—Polyphosphonic acids containing hydroxy substituents in the hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C07F9/3873—Polyphosphonic acids containing nitrogen substituent, e.g. N.....H or N-hydrocarbon group which can be substituted by halogen or nitro(so), N.....O, N.....S, N.....C(=X)- (X =O, S), N.....N, N...C(=X)...N (X =O, S)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3

Definitions

  • the acid sphingomyelinase (aSMase) is a soluble lysosomal sphingolipid hydrolase, which constitutively degrades sphingomyelin from internalized membrane fragments ( T. Kolter, K. Sandhoff, Angew. Chem. 1999, 111, 1632 ; Angew Chem Int Ed 1999, 38, 1532 ).
  • aSMase acid sphingomyelinase
  • This membrane-associated enzyme shows biochemical activity in serum and urine. Its activity is elevated in several diseases.
  • the secretory form of aSMase is believed to play an important role in signal transduction, since it alters the composition of the plasma membrane within putative sphingolipid- and cholesterol-rich membrane micro-domains.
  • 'lipid rafts' have been suggested to act as 'signalling platforms' ( K. Simons, E. Ikonen, Nature 1997, 387, 569 ) and there is significant evidence, that the cleavage of sphingomyelin to ceramide is able to dramatically alter the biophysical properties of the putative rafts ( Megha, E. London, J Biol Chem 2004, 279, 9997 ).
  • the aSMase is emerging as an important drug target in a variety of diseases. Amongst others, it has been shown that inhibition of aSMase prevents bacterial infections in a rat model of cystic fibrosis and formation of acute lung injury (ALI) elicited by endotoxin, acid instillation or platelet-activating factor (PAF). Moreover, the aSMase is essential for infection of non-phagocytotic cells with Neisseria gonorrhoea and formation of pulmonary emphysema. Pharmacological or genetic inhibition of aSMase prevents apoptosis and degeneration of liver cells in a mouse model for Wilson's disease. In addition, there are several reports that aSMase significantly contributes to the formation of atherosclerotic plugs.
  • ALI acute lung injury
  • PAF platelet-activating factor
  • Phosphatidylinositol-3,5-bisphosphate Ptdlns3,5P 2
  • this inhibitor is not suited for cell culture studies or straight forward in vivo application, because of its 5-fold negative charge and its two long fatty acid chains causing it to stack in cellular membranes.
  • this inhibitor is labile towards phospholipases A 1 , A 2 , C and D and phosphoinositide phosphatases.
  • Compounds of Formula I comprise geminal bisphosphonates and mixed phosphonate/phosphate compounds described by: wherein
  • a substituted hydrocarbon is a hydrocarbon wherein at least one hydrogen is replaced by a an atom different from hydrogen or by a group of atoms.
  • R1 is:
  • R1 is:
  • R 2 can be H, OH, CH 3 , NH 2 , or N(CH 3 ) 2 .
  • R 6 is H, OH or NH 2 , more preferably R 6 is OH or NH 2 .
  • the integer p can be from 4 to 12, preferably from 5 to 10.
  • Preferred compounds of Formula I or Formula II are the compounds:
  • Also preferred compounds are already known bisphosphonates of Formula I comprising the following compounds which have already been used for clinical applications, in particular for treatment, diagnosis and/or prophylaxis of osteoporosis:
  • compounds of Formula I and Formula II inhibit enzyme activity of aSMase in vitro and in vivo, these compounds can be used as inhibitors of aSMase.
  • a compound of Formula I or II can be used for inhibition of acid sphingomyelinase enzyme activity in vitro.
  • in vitro refers to any use or method not practised on the human or animal body. Such a use encompasses the use of compounds of Formula I or II in a cellular or cell-free assay for aSMase activity.
  • Compounds of Formula I or II may be used as a medicament, in particular as a medicament for treatment, diagnosis and/or prophylaxis of a disease associated with altered, elevated or unwanted aSMase enzyme activity.
  • aSMase enzyme activity plays a crucial role in a number of diseases.
  • Diseases which have already been associated with aSMase activity comprise e.g.:
  • NPD Niemann-Pick Disease
  • Type A and B is one of several known lysosomal storage diseases. It is a rare, recessively inherited disease caused by mutations in the gene coding for the acid sphingomyelinase (aSMase) leading to a partial loss of functional enzyme in the lysosomes.
  • aSMase acid sphingomyelinase
  • sphingomyelin a major constituent of eukaryotic plasma membranes and the substrate of acid sphingomyelinase can not be degraded, but accumulates within the lysosomes of the affected organs of NPD patients.
  • Type A or B NPD residual acid sphingomyelinase activity
  • infantile Type A aSMase activity less than 3% of normal
  • Type B NPD aSMase activity less than 6% of normal aSMase activity
  • Enzymatic activity above a threshold level of about 10% usually results in a complete or at least sufficient sphingomyelin turnover without any pathological phenotype.
  • lysosomal storage disorders like NPD Type B are likely to be protein misfolding diseases, because alterations within the active site of an enzyme normally results in a complete loss of activity.
  • a new, but very promising approach to treat lysosomal storage disorders is the use of small molecule substrate analogues or competitive inhibitors as chemical chaperones.
  • the benefit of chemical chaperones is to protect variant enzymes from being degraded by the proteasome and to facilitate their transport to the lysosomes, thereby rescuing enzymatic activity.
  • variant lysosomal enzymes produced as a consequence of an inborn genetic mutation in the aSMase gene might be active in the acid environment of the lysosomes if only they could get there.
  • Chemical chaperone mediated protection of variant enzymes occurs probably due to stabilisation of the native state fold of an otherwise misfolded enzyme by binding to its active site. Because of the very different chemical environments in the ER and in the lysosomes, some variant enzymes, which do not fold properly in the ER might retain partial or even full catalytic activity within the acidic chemical environment of the lysosomes.
  • an inhibitor of an enzyme in vitro can act as an enzyme activator in vivo.
  • potent inhibitor provides an effective chaperone, whereas less potent inhibitors require higher concentrations to achieve the same effect. This notion is most important, since potent inhibitors are expected to have therapeutic effects at lower concentrations that interact more specifically with the enzyme. By contrast, higher concentrations of moderately potent inhibitors are more likely to cross-react with other proteins.
  • Heat shock protein 70 promotes the survival of cells, e.g. cancer cells, by stabilizing lysosomes, a hallmark of stress-induced cell death. ( J. Nylandsted, M. Gyrd-Hansen, A. Danielewicz et al., J Exp Med 200 (4), 425 (2004 )). In cancer, a portion of Hsp70 translocates to the lysosomal compartment. It could be shown that Hsp70 stabilizes lysosomes by enhancing the activity of lysosomal acid sphingomyelinase.
  • aSMase The pharmacological and genetic inhibition of aSMase effectively reverts the Hsp70-mediated stabilization of lysosomes ( T. Kirkegaard et al. Nature , in revision).
  • inhibitors of aSMase sensitize cancer cells and tumours to chemo- or radiotherapy and therefore can be used in treatment, diagnosis and/or prophylaxis of cancer.
  • compounds of Formula I or II can be used in treatment, diagnosis and/or prophylaxis of infectious diseases, bacterial infections, infection with Neisseria gonnorhoeae, infections associated with cystic fibrosis, bacterial infections associated with cystic fibrosis, lung diseases, acute lung injury, acute respiratory distress syndrome, lung oedema, pulmonary emphysema, cystic fibrosis, Morbus Wilson, atherosclerosis, coronary heart disease, cardiovascular diseases, diabetes type II, depression, Alzheimer disease and/or Niemann-Pick disease and cancer.
  • a compound of Formula II is used as a medicament.
  • a compound of Formula I or II can be used for the preparation of a medicament for inhibition of acid sphingomyelinase enzyme activity.
  • a compound of Formula I or II can be used for the preparation of a medicament for treatment, diagnosis and/or prophylaxis of infectious diseases, bacterial infections, infection with Neisseria gonnorhoeae, infections associated with cystic fibrosis, bacterial infections associated with cystic fibrosis, lung diseases, acute lung injury, acute respiratory distress syndrome, lung oedema, pulmonary emphysema, cystic fibrosis, Morbus Wilson, atherosclerosis, coronary heart disease, cardiovascular diseases, diabetes type II, depression, Alzheimer disease and/or Niemann-Pick disease and cancer.
  • the present invention also refers to a method of treatment, diagnosis or prophylaxis of a disease associated with aSMase activity, comprising the administration of an effective amount of a compound of Formula I or Formula II.
  • An effective amount is an amount that yields to a measurable result with regard to treatment, diagnosis or prophylaxis of a disease associated with aSMase activity.
  • Example 1 Bisphosphonates and mixed phosphonate/phosphate compounds of Formula I and Formula II are potent and selective inhibitors of aSMase
  • Bisphosphonates are known to form bidentate complexes with Me 2+ -ions like Ca 2+ , Zn 2+ and Mg 2+ . With an additional hydroxyl or amine group, even more stable tridentate complexes can be formed. In fact, ⁇ -amino substitution leads to more stable complexes than an ⁇ -hydroxyl substitution, suggesting that aSMase inhibition also correlates with the tendency of the compounds to form complexes with the Zn 2+ residing in the reactive center of the aSMase. It is noteworthy that aSMase, both in its lysosomal and its secreted form, is a Zn 2+ -dependent enzyme.
  • the lysosomal variant is not inhibited by EDTA and not stimulated by Zn 2+ , which can be explained by abundance of Zn 2+ in the lysosomes, whereas the secreted variant is stimulated by Zn 2+ .
  • compound 7c was tested in presence of millimolar concentrations of Ca 2+ , Mg 2+ or Zn 2+ , respectively. The inhibitory activity was not significantly diminished by the metal ions.
  • Scheme 1 Synthesis of bisphosphonates.
  • Reagents and conditions a) NaH, Toluene, 60 °C, 16 h b) HCI, reflux, 16 h c) P(OMe) 3 , 0 °C, 2 h d) HP(OMe) 2 , n Bu 2 NH, 0 °C, 16 h e) H 3 PO 3 , MsOH, then PCl 3 , 90 °C, 16 h f) PCl 3 /H 3 PO 3 , 70°C, 12 h, then H 2 O, 2h
  • the aSMase inhibitor 7c was tested for any inhibitory effect on the Ser/Thr phosphatase 1 (PP1), which - like the phosphodiesterase domain of aSMase - belongs to a family of dimetal-containing phosphoesterases.
  • the PP1 enzyme was not inhibited by 7c, even at a concentration of 2 ⁇ M, which shows that this aSMase inhibitor is selective vs. PP1 (see Figure 3 ).
  • the mixed phosphate/phosphonate compounds 16 and 17 were synthesized and tested. Whereas the mixed phosphate/phosphonate compound 17 is as active as its bisphosphonate analogue 15b, the methyl ester 16 is totally inactive towards aSMase, suggesting that aSMase inhibition is dependent on the metal complexing properties of the bisphosphonates.
  • Example 2 Inhibition of aSMase activity can efficiently inhibit apoptosis
  • HepG2 liver cells were treated with dexamethasone (10 -8 M) in order to induce apoptosis, 0.1 ⁇ M of the aSMase inhibitor 7c efficiently inhibited apoptosis, as measured with a commercially-available DNA-fragmentation ELISA ( Figure 1 ).
  • Example 3 Inhibition of aSMase activity can inhibit pulmonary oedema
  • the simple bisphosphonate 7c is the most potent aSMase inhibitor found so far. It is more than 5.000fold selective against the Mg 2+ -dependent isoenzyme nSMase and selective against the dimetal-containing remote aSMase-homologue Ser/Thr protein phosphatase 1.
  • the compound which easily can be synthesized in gram-scale is also active in cell culture and efficiently protects HepG2 cells from dexamethasone-induced apoptosis.
  • Enzyme assays Crude preparations containing aSMase or nSMase were made from stripped rat brains, as described before.
  • the micellar nSMase assays using 14 C-labeled sphingomyelin as a substrate were performed as described before ( V. Wascholowski, A. Giannis, Angew. Chem., 2006, 118, 841 ; Angew Chem Int Ed 2006, 45, 827 ).
  • the fluorescent aSMase assay was performed in a 384-well-plate using the HMU-PC (6-Hexadecanoylamino-4-methylumbelliferyl- phosphorylcholine) substrate.
  • Reaction mixtures consisted of 13.3 ⁇ L HMU-PC, 13.3 ⁇ L reaction-buffer (100mM NaOAc, pH 5.2, 0.2% (w/v) Na-TC, 0.02% (w/v), 0.2% (v/v) Triton X-100) and 13.3 ⁇ L enzyme preparation. Inhibitors were added in various concentrations and the reactions were incubated for 3 hours at 37°C in a plate reader (FLUOstar OPTIMA, BMG labtech). The fluorescence of HMU (6-Hexadecanoylamino-4-methylumbelliferone) was measured (excitation 380nm, emission 460nm) in real time. Assays using the radio-labelled sphingomyelin gave the same results.
  • Apoptosis assay First, the kinetics of DNA fragmentation after dexamethasone-donation was measured in the lysate and in the supernatant, respectively. Between 6h and 8h, there was a steep increase in absorbance in the probes from the supernatant, which is typical for apoptosis (data not shown). The apoptosis assay was performed according to the manufacturer's protocol (Roche cat. No. 11585045). Briefly, cells were harvested and suspended in culture medium (2 ⁇ 10 5 cells/ml) containing BrdU labelling solution (10 ⁇ M final concentration) and plated in a 96-well cell culture dish at ⁇ 1x10 4 cells per well. After 16h, cells were washed and new media was added.
  • cells were treated with 10 -8 M of dexamethasone and 0.1 ⁇ M of 7c, respectively. After 7 hours of incubation, 100 ⁇ l of the supernatant was collected and added to a 96well plate containing immobilized anti-BrdU antibody. After incubation, removal of the supernatant and extensive washing, the secondary antibody and the TMB substrate were added and absorbance was measured at 370 nm (FLUOstar OPTIMA, BMG labtech). The experiment was performed in quintuplicate.
  • PAF-induced pulmonary edema Female Wistar rats (weight 220 to 250g) were kept on a standard laboratory chow and water ad libitum. Rat lungs were prepared, perfused and ventilated essentially as described ( S. Uhlig, E. Gulbins, Am. J. Respir. Crit. Care Med. 2008, 178, 1100 ). Briefly, lungs were perfused through the pulmonary artery at a constant hydrostatic pressure (12 cm H 2 O) with Krebs-Henseleit-buffer containing 2% albumin, 0.1% glucose and 0.3% HEPES. Edema formation was assessed by continuously measuring the weight gain of the lung.
  • platelet-activating factor causes rapid edema formation that is in part dependent on acid sphingomyelinase.
  • 7c was dissolved in buffer and added to the buffer reservoir 10 min prior to PAF (5 nMol) administration. Isolated perfused rat lungs were perfused for 30 min before 7c was added to the perfusate. 10 min later 5 nMol PAF was added as a bolus and weight gain was followed for 10 min. Data are shown as mean ⁇ SD from 4 independent experiments in each group. Statistics: 0.1 ⁇ M 7c: p ⁇ 0.01 vs PAF alone; 1 ⁇ M 7c: p ⁇ 0.01 vs. PAF alone and vs. 0.1 ⁇ M 7c /PAF (Tukey's Test).
  • PP1 assay The protein phosphatase 1 (PP1, New England Biolabs P0754L) activity was assayed in a reaction mixture of 50 ⁇ L according to the manufacturer's conditions, but containing only 1 % (500 ⁇ M) of the recommended amount of p -nitrophenylphosphate (PNPP, New England Biolabs P0757L).
  • the substrate and various inhibitor concentrations were added to the reaction buffer containing 1mM MnCl 2 , 50mM HEPES, 100mM NaCl, 0.1mM EGTA, 2mM dithiothreitol, 0.025% Tween 20 at pH 7.5.
  • the reaction was initiated by addition of PP1 (1.25U). After 6 min the reaction was quenched by addition of 10 ⁇ l of 0.5M EDTA-solution (pH 8).
  • the amount of the formed product, p-nitrophenol was determined by measuring the absorbance at 405nm (Nanodrop).
  • the control was composed as described above, including 0.2 ⁇ M 7c but with heat-denatured enzyme. All measurements were done at least in triplicate.
  • N , N -Dimethyldecanamide (1.0g, 5.02mmol) was slowly added to an initially stirred mixture of phosphorus trichloride (1.0ml, 11.4mmol) and phosphorous acid (0.42g, 5.12mmol). The mixture was heated at 70°C for 2h. After cooling the excess phosphorous trichloride was decanted off and the residue hydrolyzed by the careful addition of plenty of water. This mixture was left to stir for at least 2h, filtered, and the filtrate evaporated to dryness under reduced pressure. The precipitate was taken up in 20ml of water and heated at 100°C for 1 h, followed by filtration of the hot solution.
  • N , N -Dimethylhexanamide (1.5g, 10.6mmol) was slowly added to an initially stirred mixture of phosphorus trichloride (2.8ml, 32.2mmol) and phosphorous acid (1.15g, 14.0mmol). The mixture was heated at 70°C for 2h. After cooling the excess phosphorous trichloride was decanted off and the residue hydrolyzed by the careful addition of plenty of water. This mixture was left to stir for at least 2h, filtered, and the filtrate evaporated to dryness under reduced pressure. The precipitate was taken up in 20ml of water and heated at 100°C for 1 h, followed by filtration of the hot solution.
  • Decanoylchloride (4g, 21.0mmol) was placed in a mechanically stirred reaction flask and cooled to 0°C. Trimethylphosphite (2.60g, 21.0mmol) was added drop wise with rapid stirring (gas evolution). After addition was complete the reaction mixture was allowed to warm up at room temperature. The reaction mixture was evaporated under reduced pressure. To the colourless oil was added dimethylphosphite (1.15g, 10.5mmol) and ether (50ml), followed by an addition of di-n-butylamine (0.14g, 1.05mmol) and cooling to 0°C. The reaction mixture was allowed to warm up at room temperature and was stirring over night.

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EP09168712A 2009-08-26 2009-08-26 Bisphosphonates en tant qu'inhibiteurs d'acide sphingomyélinase Withdrawn EP2289900A1 (fr)

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Application Number Priority Date Filing Date Title
EP09168712A EP2289900A1 (fr) 2009-08-26 2009-08-26 Bisphosphonates en tant qu'inhibiteurs d'acide sphingomyélinase
PCT/EP2010/062134 WO2011023624A1 (fr) 2009-08-26 2010-08-19 Bisphosphonates comme inhibiteurs de sphingomyélinase acide
EP10759827A EP2470550A1 (fr) 2009-08-26 2010-08-19 Bisphosphonates comme inhibiteurs de sphingomyélinase acide
US13/391,328 US20120178720A1 (en) 2009-08-26 2010-08-19 Bisphosphonates as Inhibitors of Acid Sphingomyelinase
CA2771933A CA2771933A1 (fr) 2009-08-26 2010-08-19 Bisphosphonates comme inhibiteurs de sphingomyelinase acide

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Publication number Priority date Publication date Assignee Title
EP3225228A1 (fr) * 2016-03-31 2017-10-04 Ivoclar Vivadent AG Monomères hybrides acides et matériaux dentaires basés sur ceux-ci

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